Abstract
Using a DNA polymerase coupled assay and FRET (Förster resonance energy transfer)-based helicase assays, in this work, we show that a monomer of Saccharomyces cerevisiae Pif1 can unwind dsDNA (double-stranded DNA). The helicase activity of a Pif1 monomer is modulated by the nature of the 3'-ssDNA (single-stranded DNA) tail of the substrate and its effect on a Pif1-dependent re-winding activity that is coupled to the opening of dsDNA. We propose that, in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3'-ssDNA of the substrate. Interaction of DNA with this site modulates the degree to which re-winding counteracts unwinding. Depending on the nature of the 3'-tail and the length of the duplex DNA to be unwound, this activity is sufficiently strong to mask the helicase activity of a monomer. In excess Pif1 over the DNA, the Pif1-dependent re-winding of the opened DNA strongly limits unwinding, independent of the 3'-tail. We propose that, in this case, binding of DNA to the second site is precluded and modulation of the Pif1-dependent re-winding activity is largely lost.